ABSTRACT We propose to develop a system of in vivo ultrasound-mediated nucleic acid targeting for regenerative medicine. As experts in skeletal tissue regeneration, we intend to provide proof of concept in orthopedic injury models, first. Nonunion bone fractures and tendon/ligament tears are unmet clinical needs in the field of orthopedic medicine and require the development of novel therapies. Current treatments to nonunion fractures and ligament tears include autografts and allografts, which all involve serious complications and prolonged periods of healing and loss of mobility. Bone Morphogenetic Protein (BMP) gene delivery, accomplished using viruses, has been shown to induce healing of nonunion fractures in rodents and large animals. While viral vectors are the most efficient gene delivery tools, they also introduce potential risks of tumorigenic and immunogenic reactions. Nonviral vectors are considered safer for human use, albeit much less efficient for gene expression. An alternative physical method of gene transfection termed sonoporation is especially attractive for clinical applications due to the widespread use of ultrasound in the clinic today. Here, we will employ local injection of microbubbles and subsequent ultrasound imaging and therapy to achieve high efficiency local transfection. Recently we were able to demonstrate that ultrasound-based BMP-6 gene delivery yielded complete segmental bone defect repair in a mini-pig model (Science Translational Medicine, 2017). Furthermore, a similar approach was used to deliver plasmid DNA to bone tunnels created for the ligament reconstruction in a minipigs knee joints. However, currently three main hurdles prevent widespread implementation of sonoporation for orthopaedic surgery: 1. The segmental fracture site poses a unique metal- bone-soft tissue interface, which causes strong ultrasound reflections; 2. It is difficult to target the ultrasonic pulse into narrow bone tunnels for tendon/ligament graft osseointegration, and 3. The control of cavitation activity is also a hurdle and an opportunity for innovation and clinical advancements. Here we propose to rely on the extensive expertise of the Ferrara group in the field of ultrasound?controlled drug delivery to overcome the abovementioned hurdles and promote ultrasound mediated skeletal regenerative engineering to the clinic. We propose two aims: Aim 1: Engineer and test image-guided transfection for critical-size bone fractures. Goal: 1A) Develop transducer and software to sweep the US beam within a 3D volume while mapping and controlling cavitation activity. Goal: 1B) Test the efficacy of fracture healing in a pig critical-size fracture model. Aim 2: Engineer and test image-guided transfection customized for ligament/tendon bone tunnels. Goal: 2A) Develop transducer and software to direct the US beam within a bone tunnel while mapping and controlling cavitation activity. 2B) Test the efficacy of enhanced repair in a pig ligament reconstruction model. If successful, this project could be highly beneficial for numerous clinical applications beyond orthopedic targets, including cancer therapy.